이 작품은 미국 암 협회 (PF - 07-103 - 01 - CSM), 국립 보건원 (R01 GM61275 및 P30 CA042014), 백혈병과 림프종 사회, 헌츠먼 암 재단에 의해 지원되었다.

siRNA의 transfection과 셀 준비 <ol><li> 사용 각 잘하는 fibronectin (10μg/mL)의 0.5mL를 추가하여 4 잘 LabTek II chambered의 coverglass를 준비합니다. 상온에서 10~15분에 대한 부화. </li><li> 제조 업체의 지시에 따라 반대로 transfection에 대한 siRNA / Lipofectamine RNAiMAX (Invitrogen) 단지를 준비합니다. (100μL 총) 사용할 수 챔버 당 24 잘 요리를 잘 하나 권장 금액을 사용합니다. siRNA의 transfection에 대한 유사 제품 / 프로토콜은 대체 수 있습니다. </li><li> chambered의 coverglass의 우물에서 fibronectin을 제거합니다. 사용 각 잘하는 siRNA / Lipofectamine RNAiMAX 단지의 100μL를 추가합니다. </li><li> 나누어지는 20,000 히스톤 H2B - mCherry - 표현 잘 transfection 혼합물을 포함하는 당 세포를 (500μL)입니다. </li><li> 세포가 정상 세포 배양 매체 1mL와 함께 transfection 혼합물을 교체하기 전에 ~ 24 시간 동안 부화 수 있습니다. </li><li> 이미지 수집하기 전에 부화는 세포 추가 24 시간 (48시간 포스트 transfection). </li></ol> 현미경 설정 및 이미지 수집 <ol start="7"><li> 이미지 수집 설정을 세 가지 주요 부분이 있습니다 : 1) 현미경의 무대에서 맞는 37의 지속적인 습기가 환경을 유지하고 세포 배양기 ° C, 5 %의 CO 2, 2) 자동 스테이지를 갖춘 바뀌 현미경, 자동 형광 및 브라이트 셔터 및 자동 필터 바퀴, 그리고 3) 소프트웨어 무대를 통합하고 제어 패키지, 셔터, 및 필터 바퀴. 우리 이미지 설정에서, 우리는 LabTek II chambered coverglass와 함께 사용할 수 있습니다 오코 연구소에서 사용자 정의 셀 인큐베이터를 사용합니다. 무대, 셔터, 및 필터 바퀴는 Metamorph 소프트웨어에 의해 제어되는 힌트는 :. 오코 랩 무대 위로 보육 시스템은 다른 접시, 접시, 또는 coverslip 크기의 다양한 어댑터를 포함하고 있습니다. </li><li> , 미리 예열 및 equilibrated 인큐베이터의 뚜껑을 열고 어댑터에 chambered의 coverglass을 배치하고, 모델링 점토와 장소에서 개최하여 인큐베이터에서 LabTek II chambered의 coverglass 위치. 세포 배양 매체가 건조하지 않도록 chambered의 coverglass에 덮개를 둡니다. 뚜껑을 닫고 인큐베이터가 자리에 단단히 개최 있도록, 현미경 단계에있는 전체 배양기를 위치. </li><li> Metamorph를 사용하여 소프트웨어의 메뉴 막대에서 &quot;애플 리케이션 / 다차원 취득&quot;을 선택하여 실험을 설정합니다. 이것은 timelapse의 빈도와 길이를 선택할 수있는 창이 나타납니다, 각 색상에 대한 노출 시간 (경우 여러 설정하고), 번호와 무대 위치의 위치, 그리고 Z - 섹션의 수를 (경우 원하는). . 데이터 파일을 저장할 위치를 선택 힌트 : Metamorph 이외의 소프트웨어를 사용하는 경우, 기능이 약간 다를 수 있습니다하지만 전반적인 개념은 동일합니다. 마이크로, 대부분의 하드웨어 설정과 호환되는 오픈 소스 기반의 ImageJ 수집 소프트웨어는 훌륭한 대안입니다. </li><li> .이 프로토콜의 목적을 위해, 우리는 두 가지 파장 (브라이트 및 mCherry) 15 단계 위치 힌트 8 시간 동안 매 15 분 영상을 얻은 것입니다 더 나은 시간 해상도가 원하는 경우, 다음이 인수 사이의 시간 간격을 단축할 수 있으나 , 적은 무대 위치를 사용할 수 있으며, 따라서 적은 mitotic 세포는 분석을 위해 사용할 수 있습니다. 시간 간격이 계정에 필터 설정 사이를 전환할 수있는 필터 휠을에 필요한 시간을 위해 고려할 때 그것은 중요하다. </li><li> 첫째 브라이트 조명을 사용하여 전지의 분야에 초점을하여 각 파장에 대한 최적의 노출 시간을 확인합니다. . 소프트웨어에만 브라이트 채널 (이것은 각각의 인수하기 전에 각 단계의 위치에 오토포커스 소프트웨어를 수)두기에 대한 자동 초점 기능을 조정 : 우리는 석유없이도 뛰어난 해상도를 제공하는 40x 드라이 위상 대비 목표를 사용하여 . 다른 목표는 원하는 광학 해상도에 따라 사용할 수 있습니다. </li><li> mCherry 파장로 전환하여 50 밀리초 노출을 사용하여 이미지를 스냅. 좋은 이미지를 볼하는 데 필요한 최소 노출 시간을 조정합니다. 그것은 지속적인 세포 생존을 보장하기 위해 빛의 노출의 양을 제한하는 것이 필수적입니다. 일반적으로, 이것은 가장 조명 강도를 높임으로써보다 중립적인 밀도 필터 (여기 빛의 유량을 줄이기 위해)와 더 이상 카메라 노출 시간을 사용하여 얻을 수 있습니다. 사용할 수있는 추가적인 파장에 대해이 절차를 반복 힌트 :. 실험을 위해 주어진 노출에 장기 세포 생존을 확인하려면, 당신은 일시적으로 timepoint 간격을 조정하여 원하는 timelapse 이상의 세포가 살아남을 수 이미지의 총 수를 미리 볼 수 있습니다 초 분 (즉, 10 분. 10초됩니다.) 당신의 세포가 100-200 노출 (또는 살아남을 수있다면적절한 숫자) 후 노출이 괜찮습니다. 그렇지 않다면, 당신 여기 조명, 노출 시간, 또는 전체 실험을 시뮬레이션하는 이미지의 총 수를 줄일 수 있습니다. </li><li> 다음을 선택하고 셀 인구의 충분한 샘플링을 달성할 수 있도록 무대 위치의 적절한 숫자를 표시합니다. Metamorph의 위치를 기록하고 각 인수에 대해이 반환됩니다 힌트를 :. 나는 일반적으로 그들이 너무 밀도 수 있도록 많은 많은 세포의 이미지에가있는 위치를 찾으려고하지만,하지 않습니다. 또한 유사 분열을 통해 진행 세포의 대량 관찰의 좋은 기회를 얻게 될 수 있도록 충분한 위치를 선택하는 것이 중요합니다. </li><li> 모든 timelapse, 파장 노출 시간, 무대의 위치 정보가 기록된 후에는 소프트웨어가 적절하게 장비를 제어인지 확인 화면에 &quot;미리보기&quot;버튼을 선택합니다. </li><li> 당신이 만족되면, 수집을 시작하려면 화면에서 &quot;가져오기&quot;버튼을 선택합니다. 그런 다음 다시 앉아 컴퓨터와 작업을 수행하는 현미경을 허용 힌트 :. 모든 것이 제대로 작동하기 위해 인수 중 적어도 하나 둥근 통해 자동화를 관찰. </li></ol> 이미지 프로세싱 및 분석 <ol start="16"><li> 인수가 완료되면 다음 단계는 처리하기가 쉽습니다 형식으로 이미지 데이터를 전송하는 것입니다. 이 프로토콜의 목적을 위해, 나는 ImageJ 프로그램을 사용하여 이미지 montages를 생성하는 방법을 다음 Metamorph를 사용하여 동영상을 생성하는 방법을 보여 첫번째 것입니다. 어느 형식 부량 목적에 유용합니다. </li><li> 첫 번째 단계는 데이터를 검토하는 것입니다. 이렇게하려면, 메뉴 표시줄에서 &quot;애플 리케이션 / 검토 다차원 데이터&quot;를 선택하십시오. 파일의 위치를​​ 선택한 다음, &quot;보기&quot;를 선택하십시오. 이것은 개별적으로 각 단계의 위치에서 이미지의 스택을 검토하실 수 있습니다. 원하는 무대 위치를 선택한 다음 &quot;이미지로드&quot;. 당신은 데이터 프레임별로 프레임을 통해 사전하실 수 있습니다. </li><li> 세포가 유사 분열 (같은 DNA와 세포의 형태 모두에 의해 결정)을 통과하는 그 무대 위치 들어, Metamorph를 사용하여 영화와 montages를, 또는 ImageJ (NIH를 통해 무료로 가능)와 같은 다른 프로그램과 수 있습니다. </li><li> Metamorph에서 영화를 만들려고, &quot;프로세스 / 동영상 저장&quot;을 선택합니다 메뉴 표시줄 인치 당신은 어떤 사용하는 프레임의 속도와 동영상의 파일 유형을 선택하실 수 있습니다. 그런 다음 &quot;저장 무비&quot;를 선택하십시오. </li><li> 몽타주를 만들기 위해선, 그런 ImageJ 같은 다른 프로그램에서 사용할 수있는,. stk 파일로 이미지 스택을 저장합니다. </li><li> ImageJ에서 파일을 열고 당신은 유사 분열을 통해가는 세포를 찾을 때까지 프레임을 사전. . 세포 주변의 영역을 자르기와 &quot;이미지 / 복사&quot;를 선택 힌트 : 모든 프레임을 복제해야합니다. </li><li> 몽타주를하려면, &quot;이미지 / 스택 / 몬티지 만들기&quot;를 선택합니다 메뉴 표시줄 인치 여기 당신이 몽타주의 크기와 모양을 포함하고자하는 프레임 지정할 수 있으며 프레임 사이의 경계선을할지 여부. 다음을 선택하고 &quot;좋아&quot;누르십시오. .. TIF 파일로 몽타주를 저장하고 ImageJ 또는 Adobe 포토샵 하나에서 더 이상 처리를 할 힌트 : 쉽게 이미지 프로세싱을위한 8 비트 형식으로 Metamorph 사용하는 16 비트 형식의 몽타주 파일을 변경합니다. </li><li> 유사 분열의 다른 단계에서 시간을 계산하려면, 염색체가 적도 (prophase / prometaphase 시간), 프레임에 정렬되기 전까지는 염색체가 분리 시작할 때까지 t = 0 (DNA의 응축 또는 셀 라운딩의 첫 징후), 프레임의 수를 계산 결정 (metaphase 시간), 그리고 프레임 전지 (anaphase / telophase / cytokinesis) 평평하게 시작할 때까지. 각 mitotic 단계의 계산 시간은 각 프레임 사이의 시간 간격에 따라 달라집니다. </li></ol> 대표 결과 <img src="/files/ftp_upload/1878/1878fig1tmb.jpg" border="0" /> 그림 1은 히스톤 H2B - mCherry을 표현 헬라 세포 라인을 사용하여 일반적인 컨트롤 siRNA의 transfection 실험에서 결과를 보여줍니다. 이러한 방법은 후반 유사 분열 1의 타이밍에 nucleoporin Nup153에 대한 예기치 않은 역할을 노출, mitotic 타이밍을 수치 효과적으로 사용되고 있습니다. 하시기 바랍니다 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1878/1878fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다.

<ol>
	<li>Mackay, D. R., Elgort, S. W., Ullman, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleoporin+Nup153+has+separable+roles+in+both+early+mitotic+progression+and+the+resolution+of+mitosis.">The nucleoporin Nup153 has separable roles in both early mitotic progression and the resolution of mitosis.</a> Mol Biol Cell. 20, 1652-1660 (2009).</li><li>Khodjakov, A., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+division+process+in+living+tissue+culture+cells.">Imaging the division process in living tissue culture cells.</a> Methods. 38, 2-16 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li><li>Meraldi, P., Draviam, V. M., Sorger, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+checkpoints+in+the+regulation+of+mitotic+progression.">Timing and checkpoints in the regulation of mitotic progression.</a> Dev Cell. 7, 45-60 (2004).</li><li>Mora-Bermudez, F., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+structural+dynamics+of+chromosomes+in+living+cells+by+fluorescence+microscopy.">Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.</a> Methods. 41, 158-167 (2007).</li><li>Prosser, S. L., Fry, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+the+centrosome+cycle+in+mammalian+cells.">Fluorescence imaging of the centrosome cycle in mammalian cells.</a> Methods Mol Biol. 545, 165-183 (2009).</li><li>Malureanu, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BubR1+N+terminus+acts+as+a+soluble+inhibitor+of+cyclin+B+degradation+by+APC/C(Cdc20)+in+interphase.">BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase.</a> Dev Cell. 16, 118-131 (2009).</li><li>Anderson, D. J., Hetzer, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reshaping+of+the+endoplasmic+reticulum+limits+the+rate+for+nuclear+envelope+formation.">Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation.</a> J Cell Biol. 182, 911-924 (2008).</li><li>Beaudouin, J., Gerlich, D., Daigle, N., Eils, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+envelope+breakdown+proceeds+by+microtubule-induced+tearing+of+the+lamina.">Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.</a> Cell. 108, 83-96 (2002).</li><li>Marcus, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+spindle+behavior+using+confocal+microscopy.">Visualization of spindle behavior using confocal microscopy.</a> Methods Mol Med. 137, 125-137 (2007).</li><li>Steigemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+B-mediated+abscission+checkpoint+protects+against+tetraploidization.">Aurora B-mediated abscission checkpoint protects against tetraploidization.</a> Cell. 136, 473-484 (2009).</li><li>Draviam, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+screen+identifies+a+role+for+TAO1+kinase+in+spindle-checkpoint+signalling.">A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling.</a> Nat Cell Biol. 9, 556-564 (2007).</li><li>Neumann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+by+time-lapse+imaging+of+live+human+cells.">High-throughput RNAi screening by time-lapse imaging of live human cells.</a> Nat Methods. 3, 385-390 (2006).</li><li>Stegmeier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaphase+initiation+is+regulated+by+antagonistic+ubiquitination+and+deubiquitination+activities.">Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities.</a> Nature. 446, 876-881 (2007).</li></ol>

이미징 및 형광 단백질 기술의 최근 발전과 함께, 라이브 영상은 세포 분석 2 일상적인 부분이되었다. GFP의 다양한 (그리고 다른 색상 변종 3) - 태그 단백질은 광범위하게 사용할 수 없거나 구성 상대적으로 쉽고 DNA / 염색체 역학 4, 5, centrosome 중복 6, cyclin B 역학을 포함하여 세포 분열의 특징의 수를 추적하는 데 사용할 수 있습니다 7, 핵 봉투 분해와 reassembly 8, 9, mitotic 스핀들 형성 10, cytokinesis 11 여러 단계. 형광 태그의 여기 / 발광 스펙트럼 호환 때 태그 단백질은 특정 사건 사이의 협력이 평가 수 있도록, 혼자 또는 조합에서 사용할 수 있습니다. 큰 공간 및 / 또는 시간적 해상도가 / 원하는 경우, 공촛점 레이저 스캐닝, 스피닝 디스크 또는 공명 검사를 사용할 수 있는지 여부를 현미경, 그리고 계속 증가 해상도 정교한 현미경 옵션의 목록은 성장을 계속하고 있습니다. 포유 동물 세포의 유사 분열의 라이브 영상도 높은 처리량 RNAi과 작은 분자 화면 12-14에 사용하기 위해 적응되었습니다. 따라서이 기술을 사용하여 하나의 초기 실험 비교적 간단 수 있지만,이 전략을 구축하기위한 가능성은 방대합니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lipofectamine RNAiMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13778-075</td>
<td>&nbsp;</td>
<tr>
<td>Lab Tek II Chambered Coverglass (4-well)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>12-565-337</td>
<td>Additional chamber sizes are available</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F1141</td>
<td>&nbsp;</td>
<tr>
<td>Stage-top cell incubator</td>
<td>&nbsp;</td>
<td>OKO Lab</td>
<td>&nbsp;</td>
<td>Can use any appropriate chamber</td>
</tr>
<tr>
<td>Automated inverted fluorescence microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Can use any appropriate microscope</td>
</tr>
<tr>
<td>Software package</td>
<td>&nbsp;</td>
<td>Metamorph</td>
<td>&nbsp;</td>
<td>Can use any appropriate software</td>
</tr>		</tbody>
		</table>
		</div>

time-lapse imaging of mitosis after sirna transfection

세포 조직과 유사 분열하는 동안 발생하는 염색체 역학의 변화가 밀접하게 게놈 및 세포 내용의 정확한 상속을 보장하기 위해 조정됩니다. 이러한 염색체의 움직임과 같은 유사 분열의 전형적인 이벤트가, 쉽게 특정 GFP - 태그 단백질을 표현하는 포유 동물 세포 라인의 시간 경과 형광 현미경을 사용하여 개별 셀 단위로 추적할 수 있습니다. RNAi 기반의 고갈와 함께, 이것은 특정 단백질의 수준을 낮춘 후에 결함이 발생할 유사 분열의 무대 (들)을 pinpointing위한 강력한 방법으로하실 수 있습니다. 이 프로토콜에서는, 우리는 유사 분열의 타이밍에 대한 잠재적인 mitotic 규제 단백질을 파괴의 효과를 평가하기위한 기본적인 방법을 제시한다. 세포는 siRNA와 transfected 무대 위로 배양 챔버에 배치하고, 자동화된 형광 현미경을 사용하여 몇 군데 있습니다. 우리는 어떻게 아직도 이미지 montages이나 영화 중 하나를 만들기 위해 이미지 시퀀스를 처리하는 데 시간이 경과 실험을 설정하는 소프트웨어를 사용하는 방법에 대해 설명하고, 계량 및 표현 세포 라인을 사용하여 mitotic 단계의 타이밍 분석하는 방법을 mCh​​erry을 히스톤 H2B 태그. 마지막으로, 우리는 시간 경과 실험을 설계에 대한 중요한 고려 사항을 토론합니다. 이 전략은 다른 접근법을 보완하고 약물 치료를 사용하여 세포주기를 동기화 할 필요가없이 유사 분열의 인구 2) 분석과 같은 세포에서 볼 때 관찰되지 않는 운동의 변화에​​) 감도를 1의 장점을 제공합니다. 이러한 이미징 실험에서 시각 정보뿐만 아니라 유사 분열의 하위 단계가 평가 수 있습니다뿐만 아니라 외과의 세포주기 분석에서 나타나지 않을 예상치 못한 통찰력을 제공할 수 있습니다.

Watch this Scientific Journal Video about Time-lapse Imaging of Mitosis After siRNA Transfection at JoVE.com

Time-lapse Imaging of Mitosis After siRNA Transfection

여기 이미지는 기본 프로토콜을 설명하고 siRNA의 transfection 후 라이브 포유 동물 조직 배양 세포의 mitotic 타이밍을 계량.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

time-lapse-imaging-of-mitosis-after-sirna-transfection

Nanyang Technological University

Research

JoVE Journal

Biology

16.9K 조회수.  University of Utah. 여기 이미지는 기본 프로토콜을 설명하고 siRNA의 transfection 후 라이브 포유 동물 조직 배양 세포의 mitotic 타이밍을 계량.

비디오: Time-lapse Imaging of Mitosis After siRNA Transfection

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

시간 경과 현미경과 분해 굴지 모니터링

연구

생물학

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

라이브 zebrafish의 배아에서 두 광자의 axotomy하고 시간이 경과 공촛점 영상

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

에서 다세포 행동을 기록 Myxococcus 산투스</em시간 저속 Microcinematography를 사용하여> Biofilms

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

트렌스 제닉 Zebrafish의 여러 가지 빛깔의 시간 저속 영상 : 타겟의 연결을 세포 절제에 의해 활성화 떠올리 망막 줄기 세포

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

시간 경과 비디오 현미경을 이용한 세포의 무작위 마이 그 레이션의 정량 분석

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

의 장기, 고해상도 공 촛점 시간 경과 영상<em&gt; Arabidopsis 떡잎</em발아시&gt; 표피

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

마우스의 빠른 비 침습 요추 경 막내 주사를 통해 생체 siRNA의 형질과 척수에있는 유전자 넉다운에

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Intravital 시간 경과 영상에 대 한 애기 를 장착 하는 다양 한 방법을 나뭇잎합니다

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...